Priming long-lived memory CD8+ T cells is a priority for HIV vaccine development. Immunization with recombinant adeno-associated virus (rAAV) vectors has successfully generated class I MHC restricted T cell responses in mice and non-human primates. However, improvements in immunogenicity would be aided by a detailed understanding of how antigens are processed for presentation to T cells. AAV has a broad host cell range, including dendritic cells (DC) that are required to initiate T cell responses and non-professional APC like muscle. DC that are directly transduced by rAAV after i.m. immunization should process antigens through the class I MHC pathway for direct presentation to naive T cells. One the other hand high levels of antigen produced in muscle might be transferred to DC for cross-presentation to CD8+ T cells. The dominant mode of antigen presentation after AAV immunization has not been identified. It is also not clear if direct versus cross-presentation of antigens influences the composition of the memory pool as judged by T cell frequency and the repertoire of epitopes targeted. Finally, multiple receptors on the surface of DC govern their maturation to fully competent APC and determine patterns of cytokine production. Whether binding of AAV capsids to DC has functional consequences for generating memory T cell responses is not known. This project has four specific aims to: 1. Establish that DC transduced in vitro with rAAV effectively present class I epitopes directly to CD8+ T cells. A related goal is to determine if binding of rAAV capsids triggers or inhibits DC licensing for antigen presentation to CD8+ T cells. 2. Determine if memory T cells are primed in vivo when antigen expression is restricted to muscle (cross-presentation) or dendritic cells (direct presentation). 3. Compare the frequency of memory CD8+ T cells and repertoire of targeted epitopes generated by direct versus cross-presentation of antigens. 4. Identify strategies to improve antigen delivery by rAAV vectors for direct and cross-presentation to T cells.